Characterization of human adenovirus type 5 early region 1A polypeptides using antitumor sera and an antiserum specific for the carboxy terminus

Abstract

The adenovirus 5 early region 1A (ElA: 0–4.5 map units) proteins synthesized early after infection of KB cells without pretreatment of the cells with metabolic inhibitors have been studied. The E1A mRNA species of 1.1 and 0.9 kilobases (Kb) are processed from alternative 5′ splice sites to a common 3′ splice site of the same initial transcript and code for proteins of 289 and 243 amino acids in length that share common amino and carboxy terminal peptides but differ in that 46 residues of the larger polypeptide are absent from the smaller product (M. Perricaudet, G. Akusjarvi, A. Virtanen, and U. Petterson, Nature (London) 281, 694–696,1979). By immunoprecipitating labeled proteins with an antiserum directed against a synthetic pentapeptide corresponding to the carboxy terminus of the E1A proteins as predicted from the DNA sequence, we have identified four major E1A encoded species of proteins 52,000 (52K), 50K, 48.5K, and 45K Da and two minor species of 37.5K and 35K. Using mutants pm975 (which synthesizes no 0.9-Kb mRNA) (C. Montell, E. F. Fisher, M. H. Caruthers, and A. J. Berk, Nature (London) 295, 380–384,1982) and hrl (which synthesizes truncated proteins from the 1.1-Kb RNA) (R. P. Ricciardi, R. L. Jones, C. L. Cepko, P. A. Sharp, and B. E. Roberts, Proc. Nat. Acad. Sci. USA 78, 6121–6125, 1981), we have shown the 52K, 48.5K, and 37.5K species to be products of the 1.1-Kb mRNA and the 50K, 45K, and 35K species to be products of the 0.9-Kb mRNA. Only the 52K and 48.5K products of the 1.1-Kb mRNA were detected by immunoprecipitation with antitumor serum of infected cell extracts containing all the forms of the E1A proteins. This suggests that the antigenic sites recognized by our hamster antitumor sera may be determined by the 46 amino acids unique to products of the 1.1-Kb mRNA. Characterization of the E1A products synthesized in cells infected with complementation group I host range ( hr) mutants mapping in EIA showed that hrl and hr2 were defective only for the synthesis of products of the 1.1-Kb mRNA while the profiles of polypeptides from hr3, hr4, and hr5 infected cells were identical to wild type ( wt). Studies with two mutants, dl1504 and dl313, containing deletions affecting E1A sequences coding for the amino or carboxy ends of EIA proteins suggested that neither the amino terminal 15 amino acids nor the carboxy terminal 70 amino acids were necessary for the generation of the multiple species of E1A proteins that have been mapped to the individual mRNAs.